Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5

September 23, 2016

Reference

Current Issue – vol. 113 no. 45 – Manish P. Ponda, E7059–E7068, doi: 10.1073/pnas.1615671113 Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan) Manish P. Ponda^a and Jan L. Breslow^a,¹ Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. To identify potential receptors in an unbiased manner, we used ligand–receptor capture (LRC-TriCEPS) technology as a tool for detecting T-lymphocyte surface proteins that physically interact with H497–K520 (Fig. 5A) (20). Transferrin was used as a control ligand to eliminate nonspecific interactions with the TriCEPS reagent.

Abstract

TriCEPS Ligand-Receptor Capture.

The TriCEPS reagent is a proprietary trifunctional molecule with three key moieties: (i) an amine-reactive group capable of binding a peptide ligand of interest, (ii) a cross-linking group capable of bonding to oxidized glycans, and (iii) an affinity tag for downstream extraction (20, 50). These studies were performed by DualSystems Biotech. Briefly, either human H₄₉₇–K₅₂₀ or transferrin was coupled to the TriCEPS reagent. The TriCEPS–ligand complex was added to CCRF-CEM cells in complete medium in the presence or absence of zinc (100 μM), after treatment with an oxidizing reagent to oxidize surface glycoproteins. Cells were then lysed and processed as described, including LC-MS/MS analysis of captured peptides (20). Comparisons between conditions were made in triplicate to determine the relative fold enrichment of a given protein. P values were determined for pairwise comparisons and adjusted for multiple comparisons.

Author

Manish P. Ponda ^aLaboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065 Jan L. Breslow ^aLaboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065